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A robust, flow-based, microfluidic device for siRNA-mediated gene knockdown in glioblastoma spheroids

基因敲除 球体 微流控 细胞培养 细胞生物学 癌症研究 下调和上调 生物 计算生物学 基因 纳米技术 化学 材料科学 遗传学
作者
Inès Hosni,Alexander Iles,John Greenman,Mark Wade
标识
DOI:10.1142/s2737599423400054
摘要

Glioblastoma (GBM) is a deadly disease with a poor prognosis, there is therefore a crucial need for novel therapeutic targets. Current preclinical models of GBM fail to predict clinical outcomes, thus, new translationally relevant models are urgently needed for reliable therapeutic target validation. 3D spheroid culture of cancer cells has been shown to better reflect tumour biology than 2D monolayer culture, as has culturing cells in flow-based microfluidic devices, which mimic key aspects of the tumour microenvironment. Gene knockdown by siRNA is a key preclinical target validation tool, however, siRNA-mediated knockdown of cancer spheroids in microfluidic culture has not yet been demonstrated. Here we describe a simple and robust microfluidic device that can maintain GBM spheroids (U87 cells) for at least 7 days. Via RNA sequencing analysis, we demonstrate that spheroids grown in microfluidic culture are more proliferative than spheroids grown in static plate culture and downregulate genes associated with cell adhesion, potentially offering insights into the metastatic process. Comparison of target gene (PRMT2 and RAB21) knockdown using siRNA between 2D monolayer cultured cells, static spheroid culture and spheroids maintained in the microfluidic device showed that gene expression (as measured by quantitative-PCR) was significantly reduced in all culture systems. Knockdown was most efficient in cells grown in 2D monolayer culture followed by static spheroid culture, but we also demonstrate [Formula: see text] knockdown efficiency using the microfluidic device. In summary, this study describes an easy-to-use microfluidic culture platform and provides evidence that pre-clinical siRNA-mediated target validation studies will be possible in flow systems that mimic tumour physiology.
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